Project by van Breeman will interact with all the other Projects and Cores within this Botanical Center by evaluating in vitro and in vivo the hepatic metabolism, toxicity, pharmacokinetics, and blood-brain barrier permeability of active botanical compounds. This information will help provide a rational basis for the safe and effective use of botanical dietary supplements for women's health. In Specific Aim 1, the metabolism of active compounds identified in Projects by Farnsworth and Bolton and Analytical Core will be investigated in vitro using human and rat liver microsomes and hepatocytes. The specific isozymes of the human enzymes that are responsible for the formation of these metabolites will be identified and whether or not these compounds or their metabolites inhibit these enzymes will be determined. These studies will indicate the potential of these compounds to cause botanical-drug interactions. In Specific Aim 2, an ultrafiltration LC-MS-MS assay developed in this laboratory will be used to determine whether any electrophilic and possibly toxic metabolites of compounds in the botanical extracts can be formed in the liver. These studies will help predict toxicity at high doses. In addition, a new assay based on alkylation of the protein Keap1 and subsequent disruption of the Keap1-Nrf2 complex will be developed as a complementary assay. Since disruption of the Keap1-Nrf2 complex produces up-regulation of a variety of Phase II detoxification enzymes, exposure to low levels of botanical compounds that bind to Keap1 might actually produce a protective response. In Specific Aim 3, compounds in botanical extracts that are determined in Project by Bolton to have potential CNS effects will be assayed for blood-brain barrier permeability using a bovine brain epithelial cell monolayer model system. Finally in Specific Aim 4, the in vivo metabolism, bioavailability, and pharmacokinetics of active compounds will be investigated in rats and in a Phase I clinical trial in healthy post-menopausal women. These studies will provide information regarding the safety and pharmacokinetics of the active constituents in hops. Overall, these in vitro and in vivo studies of metabolism, metabolic activation, permeability, bioavailability, and pharmacokinetics will facilitate the design of future Phase II and III clinical trials of the safety and efficacy of these botanical dietary supplements.